We have been studying a novel autoantibody from a rabbit preimmune serum that immunostains myelinated nerves of the brain. Preimmune serum from a rabbit was found to contain antibodies that selectively immunostained myelin in brain sections of the rat. It also reacted with oligodendroglial cells in culture on Western blots of two-dimensional electrophoretic gels of rat cortex, this antiserum recognized a 46 kD protein with a basic pI. On Western blots of whole mouse brain homogenates, the autoantibody strongly stained a component that migrated to the same position as 2-, 3-cyclic nucleotide 3-phosphodiesterase (CNPL) band. Although the staining of the CNP1 was more intense with the autoantibody than that with a well characterized anti-CNP antiserum that stained both forms of CNP, there was no staining of CNP2. Comparison of the immunostaining of homogenate and myelin samples with the autoantibody and the known anti-CNP antiserum showed a similar enrichment of the stained CNP1 band in the myelin. This autoantibody may be useful in establishing the structural difference between CNP1 and CNP2, and for demonstrating possible differences in the localization of the two forms by immunohistochemistry. The presence of this protein within myelin may help identify possible cellular mechanisms of myelination, and the autoimmune serum might be useful in structural studies of myelin.